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Chapter 4 Glycosylation Precursors

Chapter 4 Glycosylation Precursors. Ya -Min Chi. Intro. Glycosylation requires activation of monosaccharides to nucleotide sugar. Nucleotide sugar is the high-energy donor form. Activated Form. Glucose Transporters. Three types Energy-independent diffusion transporter

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Chapter 4 Glycosylation Precursors

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  1. Chapter 4 Glycosylation Precursors Ya-Min Chi

  2. Intro • Glycosylation requires activation of monosaccharides to nucleotide sugar. • Nucleotide sugar is the high-energy donor form.

  3. Activated Form

  4. Glucose Transporters • Three types • Energy-independent diffusion transporter • e.g. Hexose transporter • Energy-dependent transporter • e.g. Sodium-dependent glucose transporter (SGLT) • Transporter that couple ATP phosphorylation with sugar import. • e.g. found in bacateria

  5. Mannose & Fucose Transport • Mannose • Energy-dependent transporter • Near border of enterocytes • Surface of kidney tubule epithelial • Energy-independent facilitated diffusion • Surface of many mammalian cells • Fucose • Found in several type of mammalian cells • Km-250µM

  6. Intracellular Source of Monosaccharides • Salvage • from glycoconjugates • Degradation at low pH in the lysosomes • Lysosomal carriers • Neutral sugar carrier • Km-50-75mM • N-acetylhexosamine carrier • Km-4mM • Acidic sugar carrier • Km-300-550µM

  7. Activation and Interconversion • Glycogen • Glycogenin • Glycogen phosphorylase • From UDP-Glc • Glucose • Building block of other sugar • Glycogen • Glucosylceramide • Dolichol-P-glucose

  8. Glucuronic Acid • UDP-GlcA • Synthesized from UDP-GlC • For • GAG biosynthesis • Some N-/O-linked glycans and glycosphingolipids • Iduronic Acid • C-5 epimer of glucuronic acid • Found in GAGs, dermatan sulfate, heparan sulfate, and heparin • Created from epimerization of GlcA

  9. Xylose • UDP-Xyl • Created from decarboxylation of UDP-GlcA • Initiate GAG synthesis • Mannose • Use for multiple types of glycan • GDP-Man • Primary activated donor • form Lipid-linked oligosaccharide on ER • Form dolichol-P-mannose in the ER membrane • Fucose • GDP-Fuc • From GDP-Man or directly from fucose

  10. Galactose • UDP-Gal • From Gal or UDP-Glc • N-Acetlyglucosamine • UDP-GlcNAc • From fructose or GlcNAc • N-Acetlygalactosamine • UDP-GalNAc • From GalNac or epimerization of UDP-GlcNAc • Sialic Acids • CMP-Sia • Many ways to obtain • Mostly occur in Golgi

  11. Bacteria and Plant • Animal • Fucose • The only deoxyhexoes in animal cell glycan • Bacteria and Plant • Varieties of sugars • Deoxysugars • From oxidation of C-4 • Deoxyaminosugars • Amino sugar- from keto sugar with addition of amino group from glutamine • Branched-chain sugars

  12. Nucleotide Sugar Transporters • Activated sugar must be transport • Needed in ER and Golgi • Negatively charged • Antiporters • Energy-independent • Organelle specific • Location correspond to glycosyltranferases’ location • Km-1-10µM • Export nucleoside monophosphates

  13. Control of Nucleotide Sugar Levels

  14. Other Modification

  15. Carrier Lipids • Transport sugar for glycosylation • e.g. • Undecaprenyl-P • Dolichol-P

  16. Chapter 5Glycosyltransferasesand Glycan-processing Enzymes

  17. Intro • Glycosyltransferases • Transport sugar from nucleotide sugar to substrate • Variety of enzymes • Glycosidase • Enzyme that break down glycosidic bond • For the formation of N-glycans • Other enzymes • e.g. sulfotransferases

  18. Glycosyltransferase Specificity • “one enzyme-one linkage” • Exception • some enzymes can produce the same linkage • e.g. human fucosyltransferases III-VII • Some have two separate active sites

  19. Protein/Glycoprotein Acceptors • Specificity of glycosylation reaction • Polypeptide chain of acceptor • Folding of protein acceptor

  20. GlycosyltransferaseSequence Families • Represent 1-2% of the genome • 30,000+ sequence • 90 families • 29 families’ structures have been determined

  21. Folding type • Two major types of of folding • GT-A • One single domain • Contain Rossmann fold • Contain DXD motif (asp-any residue-asp) • Metal-ion dependent • GT-B • Two domains • One is carboxy-termminal • Metal-ion independent

  22. Catalytic Mechanisms • Through either inversion or retention of stereochemistry at the anomeric carbon. inversion

  23. Kinetic Mechanisms • Bi Bi sequential kinetic mechanism • Donor substrate is bind first • Acceptor is release first

  24. Other Enzymes • Sulfotransferases • Cytoplasm and the Golgi • For glycosaminylglycans • Embryological development and physiology • For L-selectin ligand • Trafficking of lymphocytes • Use PAPS as sulfate donor

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